In this paper, first strand cdna of 3abc gene was synthesized using template rna extracted from cells infected with fmdv. the complete 3abc gene about isoobp was amplified by pcr and ligated into pgem - t easy vector. after transforming e. coli dh5 a, ampicillin resistant colonies were isolated and plasmid dna was prepared and analyzed by restriction analysis and pcr. presence of the full length 3abc gene was verified by nucleotide sequence analysis and the plasmid containing the expected sequence was named as pgem - 3abc. comparing the aquired sequence of 3abc with that of reference strains, the homology is more than 99 percent. the pgem - 3abc was digested with sal i and bgl ii and ligated into xho i and bgl ii - digested expression vector ptriex - 4 neo. lt was identified by restriction analysis and pcr and sequencing that this fragment had a 17bp deletion hi the nucleotide sequence 708bp of 3abc gene, which happened to form a terminator codon behind 3ab gene, but it contained the complete open reading frame ( orf ) of 3ab gene. positive clones were selected and induced with lmmol / l isopropyl - d - galactoside ( iptg ), bacteria were detected by sds - page and western blotting after properly treated. the results showed that the 3ab gene expressed successfully in e. coli and 33. 5ku fusion protein can be recognized by the positive bovine serum of fmdv. the amount of target protein is over 26 % of the total bacteria protein by gel thin layer scanning analysis
擴增產物連接到pgem - teasy載體中,轉化大腸桿菌dh5菌株,篩選氨芐青霉素抗性菌落,提取質粒經酶切鑒定、 pcr分析以及確證性
測序證明,所克隆的1500bp左右的片段含有完整的3abc基因,與國外參考
序列相比,同源性在99以上。將重組質粒pgem - 3abc和表達載體ptriex - 4neo分別用sal和bgl與xho和bgl消化后,亞克隆3abc基因至原核表達載體ptriex - 4neo中,通過酶切鑒定、 pcr擴增以及
序列分析,發現克隆到ptriex - 4neo載體上的片段於3abc基因708bp處出現了17bp的缺失,碰巧在3ab基因后形成一終止密碼子,但3ab基因的閱讀框架完整,選出含有3ab基因完整閱讀框架的陽性克隆,用iptg誘導表達,收集菌液進行sds - page電泳、 westernblotting分析,結果表明, 3ab基因在大腸桿菌中成功表達,其表達產物為分子量33 . 5ku的融合蛋白,並能被口蹄疫病毒陽性血清識別。經薄層掃描分析,表達量占總蛋白量的26以上。
Its sequence is consistent with the reported apoes sequence in the gene bank
; cdna片段克隆
測序,其
序列與基因庫中己報道的人apoea
序列一致。
Cloning and expression in e. coli of lactase. specific primers were designed according to the sequence of the beta - galactosidase gene from kluyveromyces lactis. klac gene was amplified by pcr and subsequently cloned
乳糖酶基因的克隆及原核表達以乳酸克魯維斯酵母( kluyveromyceslactis )菌株k538的基因組dna為模板,設計引物,利用pcr獲得乳糖酶基因klac ,經dna
測序驗證,得到克隆t1549 。
Then the pcr product was purified, ligated into pgem - t vector by ta cloning
篩選陽性克隆,
測序,大量制備
序列完全正確的質粒。
The nucleoprotein ( n ) gene of three strains and glycoprotein ( g ) gene of 119, gxbm were amplified by reverse transcription - polymerase chain reaction ( rt - pcr ), respectively
對三株的n基因和119 、 gxbm株的g基因進行了rt - pcr擴增、克隆和
測序。
Molecular cloning of 5 ' flanking region of ovine keratin associated protein 6 - 1 gene and comparison of the sequences
端調控區的分子克隆及
測序結果比較
The sequence of hntx - v had been determined by 491 sequencer : nh2 - eclgfgkgcnpsdqccksanlvcsrkhrwckyei - cooh, in which there were 35 amino acid residues and six cys. the toxin could block neuromuscular transmission in an isolated mouse phrenic nerve diaphragm preparation. hntx - v is a natural mutant of hntx - iv, for there is only one different residue ( ala20ser )
Hntx -經491
測序儀
測得其氨基酸組成為: nh2 - eclgfgkgcnpsdqccksanlvcsrkhrwckyei - cooh ,含35個氨基酸殘基, 6個半胱氨酸全部參與了二硫鍵的形成,該毒素對小鼠膈神經-膈肌標本有阻遏作用。
Using a pair of degenerate primers based on the conservative region, hmg - box, of human sry gene, tow different fragments of sox gene, essox3 and essox22 were amplified from female and male eriocheir sinensis, the sequence results indicated that essox3 and essox22 shown high homology to human sox genes, and the identities to human sox genes in dna sequence and amino acid sequence are 84 % 、 85 % and 97 % 、 81 %, respectively. it might be concluded that sox gene was highly conservative in phylogenesis
二、研究論文1 、參照人sry基因hmg - box保守區的
序列,設計一對兼并引物, pcr擴增了中華絨螯蟹的sox基因,並對擴增產物進行了克隆和
測序。結果在雌雄個體中篩選出兩個不同的sox基因essox3和essox22 ,其dna
序列和編碼的氨基酸
序列與人相應sox基因的相似性分別為84 % 、 85 %和97 % 、 81 % ,顯示該基因在進化上具高度的保守性。
Ghrelin regulates pituitary growth hormone ( gh ) secretion. in the study, we obtained the cdna sequences of preproghrelin and the cdna encoding the ghrelin of duck, goose and emu by using rt - pcr and 3 " - race methods. and then deduced the amino acid sequences of preproghrelin and ghrelin in duck. goose and emu. sense and antisense primers were designed on the basis of chicken ghrelin cdna sequence ( dest accession no. ab075215, 836bp ) and proventriculus cdna was performed as the template
Ab075215 , 836bp )設計引物,以鴨、鵝、鴯鶓腺胃cdna為模板,通過rt - pcr及3 - race的方法,將各個產物經過回收,再經連接轉化,克隆
測序,拼接后得到鴨、鵝、鴯鶓preproghrelin基因的cdna
序列及ghrelin的cdna
序列,並由此推
測出preproghrelin及ghrelin的氨基酸
序列。
Cloning of rhesus monkey tpa cdna and its expression in cho
測序與真核表達
The sucking mouse brain were inoculated with mdj - 01 strain to make electron microscopic examination, results showed that the virus was a spheral particle with membran which had a diameter of about 40 nm. by indirect fluorescent antibody test mdj - 01 strain was identified with tbev. a part of region encoding e protein was expanded by rt - pcr and sequenced. the nucleotide sequences of two strain viruses were compared with sequences in genbankjsequence homology analyses revealed mdj - 01 strain and senzhang strain had the highest homology with tbev oshima5 - 10, respectively, which were 95 %, 94 %. mdj - 01 strain was identified with tbev again
應用間接免疫熒光試驗進行血清學鑒定,結果表明mdj - 01株為tbev 。通過rt - pcr技術擴增部分e蛋白
序列並
測序,在genbank上進行同源性比較,發現mdj - 01株和森張株與tbevoshima5 - 10株的同源性最高,分別為94 、 95 ,從分子生物學水平上進一步證明mdj - 01株病毒為tbev 。在鑒定的基礎上,本實驗對兩株病毒進行了核苷酸全
序列
測定。
By transformation with the genes. plant disease biocontrol bacteria bacillus subtil is aplls and b. megaterium ap25 were isolated from wheat field soils collected from south australia and tai an. enzyme activity analysis on chitin agar and abp media showed that b. subtilis aplls secreted chitinase and b. megaterium ap25 secreted endoglucanase, respectively
測序后在genebank上進行序列比較,該基因片段同編號為2634966的枯草芽孢桿菌全序列的2599451到2812870 (功能未知)有85的同源性,但同已發表的13種幾丁質酶的基因(包括枯草芽孢桿菌幾丁質酶基因)的同源性很低,只有30 。